Peptides and combination of peptides for use in immunotherapy against cancers

ABSTRACT

The present description relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present description relates to the immunotherapy of cancer. The present description further relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T-cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/309,107, filed 16 Mar. 2016, and Great Britain Application No.1604490.1, filed 16 Mar. 2016, the content of each of these applicationsis herein incorporated by reference in their entirety.

This application also is related to PCT/EP2017/055973 filed 14 Mar.2017, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-064001_ST25.txt” createdon 14 Mar. 2017, and 2,427 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

FIELD

The present description relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentdescription relates to the immunotherapy of cancer. The presentdescription further relates to tumor-associated T-cell peptide epitopes,alone or in combination with other tumor-associated peptides that canfor example serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT-cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present description further relates to the use of the above peptidesfor the generation of specific T-cell receptors (TCRs) binding totumor-associated antigens (TAAs) for targeting cancer cells, thegeneration of T-cells expressing same, and methods for treating cancersusing same. The novel peptide sequences and their variants derived fromHLA class I molecules of human tumor cells can be used in vaccinecompositions for eliciting anti-tumor immune responses, or as targetsfor the development of pharmaceutically/immunologically active compoundsand cells. Preferred is a peptide that has the amino acid sequenceKIQEILTQV (SEQ ID NO:1).

BACKGROUND OF THE INVENTION

Gastric cancer is a disease in which malignant cells form in the liningof the stomach. Stomach or gastric cancer can develop in any part of thestomach and may spread throughout the stomach and to other organs;particularly the esophagus, lungs and the liver. Stomach cancer is thefourth most common cancer worldwide with 930,000 cases diagnosed in2002. It is a disease with a high death rate (˜800,000 per year) makingit the second most common cause of cancer death worldwide after lungcancer. It is more common in men and occurs more often in Asiancountries and in developing countries.

It represents roughly 2% (25,500 cases) of all new cancer cases yearlyin the United States, but it is more common in other countries. It isthe leading cancer type in Korea, with 20.8% of malignant neoplasms. InJapan gastric cancer remains the most common cancer for men. Each yearin the United States, about 13,000 men and 8,000 women are diagnosedwith stomach cancer. Most are over 70 years old.

Stomach cancer is the fourth most common cancer worldwide, after cancersof the lung, breast, and colon and rectum. Furthermore, stomach cancerremains the second most common cause of death from cancer. The AmericanCancer Society estimates that in 2007 there were an estimated onemillion new cases, nearly 70% of them in developing countries, and about800,000 deaths.

Tremendous geographic variation exists in the incidence of this diseasearound the world. Rates of the disease are highest in Asia and parts ofSouth America and lowest in North America. The highest death rates arerecorded in Chile, Japan, South America, and the former Soviet Union.

Gastric cancer is often diagnosed at an advanced stage, becausescreening is not performed in most of the world, except in Japan (and ina limited fashion in Korea) where early detection is often achieved.Thus, it continues to pose a major challenge for healthcareprofessionals. Risk factors for gastric cancer are Helicobacter pylori(H. pylori) infection, smoking, high salt intake, and other dietaryfactors. A few gastric cancers (1% to 3%) are associated with inheritedgastric cancer predisposition syndromes. E-cadherin mutations occur inapproximately 25% of families with an autosomal dominant predispositionto diffuse type gastric cancers. This subset of gastric cancer has beentermed hereditary diffuse gastric cancer. 12 It may be useful to providegenetic counseling and to consider prophylactic gastrectomy in young,asymptomatic carriers of germ-line truncating

The wall of the stomach is made up of 3 layers of tissue: the mucosal(innermost) layer, the muscularis (middle) layer, and the serosal(outermost) layer. Gastric cancer begins in the cells lining the mucosallayer and spreads through the outer layers as it grows. Four types ofstandard treatment are used. Treatment for gastric cancer may involvesurgery, chemotherapy, radiation therapy or chemoradiation. Surgery isthe primary treatment for gastric cancer. The goal of surgery is toaccomplish a complete resection with negative margins (R0 resection).However, approximately 50% of patients with locoregional gastric cancercannot undergo an R0 resection. R1 indicates microscopic residual cancer(positive margins); and R2 indicates gross (macroscopic) residual cancerbut not distant disease. Patient outcome depends on the initial stage ofthe cancer at diagnosis (NCCN Clinical Practice Guidelines inOncology™).

The 5-year survival rate for curative surgical resection ranges from30-50% for patients with stage II disease and from 10-25% for patientswith stage Ill disease. These patients have a high likelihood of localand systemic relapse. Metastasis occurs in 80-90% of individuals withstomach cancer, with a six month survival rate of 65% in those diagnosedin early stages and less than 15% of those diagnosed in late stages.

Gliomas are brain tumors originating from glial cells in the nervoussystem. Glial cells, commonly called neuroglia or simply glia, arenon-neuronal cells that provide support and nutrition, maintainhomeostasis, form myelin, and participate in signal transmission in thenervous system. The two most important subgroups of gliomas areastrocytomas and oligodendrogliomas, named according to the normal glialcell type from which they originate (astrocytes or oligodendrocytes,respectively). Belonging to the subgroup of astrocytomas, glioblastomamultiforme (referred to as glioblastoma hereinafter) is the most commonmalignant brain tumor in adults and accounts for approx. 40% of allmalignant brain tumors and approx. 50% of gliomas (CBTRUS, 2006). Itaggressively invades the central nervous system and is ranked at thehighest malignancy level (grade IV) among all gliomas. Although therehas been steady progress in their treatment due to improvements inneuroimaging, microsurgery, diverse treatment options, such astemozolomide or radiation, glioblastomas remain incurable (Burton andPrados, 2000). The lethal rate of this brain tumor is very high: theaverage life expectancy is 9 to 12 months after first diagnosis. The5-year survival rate during the observation period from 1986 to 1990 was8.0%. To date, the five-year survival rate following aggressive therapyincluding gross tumor resection is still less than 10% (Burton andPrados, 2000).

The onset of colorectal cancer is the result of interactions betweeninherited and environmental factors. In most cases adenomatous polypsappear to be precursors to colorectal tumors; however the transition maytake many years. The primary risk factor for colorectal cancer is age,with 90% of cases diagnosed over the age of 50 years. Other risk factorsfor colorectal cancer according to the American Cancer Society includealcohol consumption, a diet high in fat and/or red meat and aninadequate intake of fruits and vegetables. Incidence continues to rise,especially in areas such as Japan, where the adoption of westernizeddiets with excess fat and meat intake and a decrease in fiber intake maybe to blame. However, incidence rates are rising not as fast aspreviously which may be due to increasing screening and polyp removal,thus preventing progression of polyps to cancer.

As in most solid tumors, first line treatment is surgery, however, itsbenefits remain confined to early-stage patients, yet a significantproportion of patients is diagnosed in advanced stages of the disease.For advanced colorectal cancer chemotherapy regimens based onfluorouracil-based regimens are standard of care. The majority of theseregimens are the so-called FOLFOX (infusional 5-FU/leucovorin plusoxaliplatin) and FOLFIRI (irinotecan, leucovorin, bolus andcontinuous-infusion 5-FU) protocols.

The introduction of third-generation cytotoxics such as irinotecan andoxaliplatin has raised the hope of significantly improving efficacy, butprognosis is still relatively poor, and the survival rate generallyremains at approximately 20 months in metastatic disease and, as aresult, the unmet needs in the disease remain high.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) can becategorized into the following groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T-cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T-cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides. MHC class Imolecules can be found on most nucleated cells. They present peptidesthat result from proteolytic cleavage of predominantly endogenousproteins, defective ribosomal products (DRIPs) and larger peptides.However, peptides derived from endosomal compartments or exogenoussources are also frequently found on MHC class I molecules. Thisnon-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997). MHCclass II molecules can be found predominantly on professional antigenpresenting cells (APCs), and primarily present peptides of exogenous ortransmembrane proteins that are taken up by APCs e.g., duringendocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positiveT-cells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T-cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T-cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T-cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T-cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g., CTLs, natural killer (NK) cells,macrophages, and granulocytes.

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006). Elongated (longer) peptides of the description can function asMHC class II active epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killerT-cells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T-cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ). There is evidencefor CD4 T-cells as direct anti-tumor effectors (Tran et al., 2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T-cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T-cells bearingspecific T-cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e., copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g., in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Therefore, TAAs are a starting point for the development of a T-cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T-cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the description it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T-cell can be found. Such afunctional T-cell is defined as a T-cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T-cell”).

In case of targeting peptide-MHC by specific TCRs (e.g., soluble TCRs)and antibodies or other binding molecules (scaffolds) according to thedescription, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

Thus, in view of the above and similar situations in other camcers,there remains a need for new efficacious and safe treatment option forgastric cancer, prostate carcinoma, oral cavity carcinomas, oralsquamous carcinoma (OSCC), acute myeloid leukemia (AML), H.pylori-induced MALT lymphoma, colon carcinoma/colorectal cancer,glioblastoma, non-small-cell lung cancer (NSCLC), cervical carcinoma,human breast cancer, prostate cancer, colon cancer, pancreatic cancers,pancreatic ductal adenocarcinoma, ovarian cancer, hepatocellularcarcinoma, liver cancer, brain tumors of different phenotypes, leukemiassuch as acute lymphoblastic leukemia (ALL), lung cancer, Ewing'ssarcoma, endometrial cancer, head and neck squamous cell carcinoma,epithelial cancer of the larynx, oesophageal carcinoma, oral carcinoma,carcinoma of the urinary bladder, ovarian carcinomas, renal cellcarcinoma, atypical meningioma, papillary thyroid carcinoma, braintumors, salivary duct carcinoma, cervical cancer, extranodal T/NK-celllymphomas, Non-Hodgkins Lymphoma and malignant solid tumors of the lungand breast and other tumors, optimally enhancing the well-being of thepatients without using chemotherapeutic agents or other agents which maylead to severe side effects.

WO 2015/187040 relates to amino sphingoglycolipid analogues and peptidederivatives thereof, compositions comprising these compounds and methodsof treating or preventing diseases or conditions using such compounds,especially diseases or conditions relating to cancer, infection, atopicdisorders, autoimmune disease or diabetes, KIQEILTQV is SEQ ID NO:377disclosed as a peptide that contains within its sequence one or moreepitopes that bind to MHC molecules and induce T cell responses.

US 2012-0308590 discloses KIQEILTQV (SEQ ID NO:3) as belonging to IMP-3552-560 (insulin-like growth factor II mRNA binding protein 3) for lungcancer and esophageal cancer treatment. Similarly, Tomita, Y. et al.disclose the peptide as derived from IMP-3.

US 20110142919 discloses KIQEILTQV (SEQ ID NO:409) as derived fromPutative RNA binding protein KOC and identified by MS.

Dutoit et al. (2012) disclose KIQEILTQV (NP_006538) for glioblastomatreatment.

WO 2007/150077 discloses immunogenic peptides isolated from ovariancancer samples. The peptide IGF2BP3-001 is SEQ ID No: 158.

BRIEF SUMMARY OF THE INVENTION

In an aspect, the present description relates to a peptide comprising anamino acid sequence consisting of SEQ ID NO:1 or a variant sequencethereof which is at least 65%, preferably at least 77%, and morepreferably at least 85% homologous (preferably at least 75% or at least85% identical) to SEQ ID NO:1, wherein said variant binds to MHC and/orinduces T-cells cross-reacting with said peptide, or a pharmaceuticallyacceptable salt thereof, and wherein said peptide is not the underlyingfull-length polypeptide. Furthermore, said peptide is not an IMP-3and/or KOC-derived antigen.

The present description further relates to a peptide of the presentdescription comprising a sequence consisting of SEQ ID NO:1 or a variantthereof, which is at least 65%, preferably at least 75%, and morepreferably at least 85% homologous (preferably at least 75% or at least85% identical) to SEQ ID NO:1, wherein said peptide or variant thereofhas an overall length of between 8 and 100, preferably between 8 and 30,and most preferably of between 8 and 14 amino acids, wherein saidpeptide or variant binds to MHC and/or induces T-cells cross-reactingwith said peptide, or a pharmaceutically acceptable salt thereof.

The peptide according to the present description, is the peptideaccording to SEQ ID NO: 1, KIQEILTQV as well as variants thereof asdescribed herein.

The present description furthermore generally relates to the peptidesaccording to the present description for use in the treatment ofproliferative diseases, such as gastric cancer, prostate carcinoma, oralcavity carcinomas, oral squamous carcinoma (OSCC), acute myeloidleukemia (AML), H. pylori-induced MALT lymphoma, coloncarcinoma/colorectal cancer, glioblastoma, non-small-cell lung cancer(NSCLC), cervical carcinoma, human breast cancer, prostate cancer, coloncancer, pancreatic cancers, pancreatic ductal adenocarcinoma, ovariancancer, hepatocellular carcinoma, liver cancer, brain tumors ofdifferent phenotypes, leukemias such as acute lymphoblastic leukemia(ALL), lung cancer, Ewing's sarcoma, endometrial cancer, head and necksquamous cell carcinoma, epithelial cancer of the larynx, oesophagealcarcinoma, oral carcinoma, carcinoma of the urinary bladder, ovariancarcinomas, renal cell carcinoma, atypical meningioma, papillary thyroidcarcinoma, brain tumors, salivary duct carcinoma, cervical cancer,extranodal T/NK-cell lymphomas, Non-Hodgkins Lymphoma and malignantsolid tumors of the lung and breast and other tumors.

Preferred is the use in non-small cell lung cancer, small cell lungcancer, renal cell cancer, brain cancer (e.g., glioblastoma,neuroblastoma), gastric cancer, colorectal cancer, hepatocellularcancer, head and neck cancer, pancreatic cancer, prostate cancer,leukemia, breast cancer, Merkel cell carcinoma, melanoma, ovariancancer, urinary bladder cancer, uterine cancer, gallbladder and bileduct cancer and esophageal cancer. More preferred is the use inpancreatic cancer, hepatocellular cancer, gastric cancer, and/orcolorectal cancer.

Particularly preferred is the peptide—alone or in combination—accordingto the present description consisting of SEQ ID NO:1. More preferred isthe peptide—alone or in combination—consisting of SEQ ID NO:1, and theiruses in the immunotherapy of gastric cancer, prostate carcinoma, oralcavity carcinomas, oral squamous carcinoma (OSCC), acute myeloidleukemia (AML), H. pylori-induced MALT lymphoma, coloncarcinoma/colorectal cancer, glioblastoma, non-small-cell lung cancer(NSCLC), cervical carcinoma, human breast cancer, prostate cancer, coloncancer, pancreatic cancers, pancreatic ductal adenocarcinoma, ovariancancer, hepatocellular carcinoma, liver cancer, brain tumors ofdifferent phenotypes, leukemias such as acute lymphoblastic leukemia(ALL), lung cancer, Ewing's sarcoma, endometrial cancer, head and necksquamous cell carcinoma, epithelial cancer of the larynx, oesophagealcarcinoma, oral carcinoma, carcinoma of the urinary bladder, ovariancarcinomas, renal cell carcinoma, atypical meningioma, papillary thyroidcarcinoma, brain tumors, salivary duct carcinoma, cervical cancer,extranodal T/NK-cell lymphomas, Non-Hodgkins Lymphoma and malignantsolid tumors of the lung and breast and other tumors.

The IGF2BP1 gene encodes a member of the insulin-like growth factor 2mRNA-binding protein family. The protein encoded by this gene containsfour K homology domains and two RNA recognition motifs. It functions bybinding to the mRNAs of certain genes, including insulin-like growthfactor 2, beta-actin and beta-transducin repeat-containing protein, andregulating their translation. Two transcript variants encoding differentisoforms have been found for this gene. The IGF2BP1 gene maps tochromosome 17q21.32. A 580 kb microdeletion in this locus is associatedwith mental retardation, microcephaly, cleft palate and cardiacmalformation (Rooryck et al., 2008).

IGF2BP1 is over-expressed in a large variety of different cancerentities. In breast cancer, moderate (30%) and high (5%) IGF2BP1 geneamplification as well as amplification-independent over-expression ofIGF2BP1 has been detected (Doyle et al., 2000; loannidis et al., 2003).The colorectal cancer study, performed by Ross and colleagues, revealedIGF2BP1 over-expressed in the majority of colorectal cancer specimens(81%), whereas it was scarce or absent from normal colon tissue (Ross etal., 2001). Ioannidis and colleagues provide evidence for IGF2BP1expression in a panel of cancer types, including colon cancer (1/1),prostate cancer (1/4), breast cancer (3/4), different sarcoma (24/33),monomyelocytic leukemia (1/2), 12/24 malignant neuroepithelial tumors,2/5 benign neuroepithelial tumors and 4/15 non-small cell lungcarcinomas (loannidis et al., 2001; loannidis et al., 2004).Furthermore, IGF2BP1 was shown to be over-expressed in adenocarcinomaand low malignant potential tumors of the ovary (Gu et al., 2004) andovarian serous carcinoma effusions (Davidson et al., 2014) as well as inhepatocellular cancer (2/7 patients) (Himoto et al., 2005; Gutschner etal., 2014), choriocarcinoma (Hsieh et al., 2013), neuroblastoma (Bell etal., 2015) and rhabdomyosarcoma (Faye et al., 2015). Up-regulation ofIGF2BP1 expression has further been described in different testicularneoplasias, like pre-invasive testicular carcinoma in situ, classicaland spermatocytic seminoma and undifferentiated embryonal carcinoma(Hammer et al., 2005). In acute lymphoblastic leukemia, IGF2BP1over-expression appears to be characteristic for the subgroup ofETC6/RUNX1-positive tumors (Stoskus et al., 2011). In malignantmelanoma, increased expression of IGF2BP1 was shown to be associatedwith over-activity of the Wnt/beta-catenin signaling pathway (Elcheva etal., 2008). IGF2BP2 over-expression in basal cell carcinoma correlateswith activation of Wnt as well as Hedgehog signaling (Noubissi et al.,2014).

High IGF2BP1 levels appear to be associated with poor prognosis indifferent cancer entities. IGF2BP1 over-expression was shown to besignificantly correlated with reduced recurrence free and overallsurvival in ovarian cancer patients (Gu et al., 2004; Kobel et al.,2007). Furthermore, positive immunostaining of IGF2BP1 correlated withtumor size, non-well-differentiated tumor grade and poor prognosis inpatients with lung cancer (Kato et al., 2007). In colorectal cancer,IGF2BP1 expression was found to be associated with increased metastasisformation and recurrence as well as with shorter survival times(Dimitriadis et al., 2007). Over-expression of IGF2BP1 is associatedwith poor prognosis in hepatocellular cancer (Zhou et al., 2015b) andwith lower overall patient survival in neuroblastoma (Bell et al.,2015). IGF2BP1 hyper-methylation was shown to be associated with anaggressive disease phenotype in meningioma (Vengoechea et al., 2013).

IGF2BP1 expression was shown to be associated with advanced diseasestages in ovarian cancer, colorectal cancer and neuroblastoma. Koebeland colleagues report preferential detection of high IGF2BP1 expressionlevels in high-grade and late stage ovarian cancer specimens (Kobel etal., 2007). In colorectal cancer, frequency and intensity of IGF2BP1staining were shown to increase with progression of the disease to lymphnode metastases. High levels of IGF2BP1 protein were detected in 97% ofcolorectal cancer lymph node metastases and the expression level inprimary cancers correlated well with the presence of lymph nodemetastases (Vainer et al., 2008). In neuroblastoma, IGF2BP1 expressionwas associated with stage 4 cancers (Bell et al., 2015).

The present description furthermore relates to peptides according to thepresent description that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongated(longer) form, such as a length-variant—MHC class-II.

The present description further relates to the peptides according to thepresent description wherein said peptides (each) comprise, consist of,or consist essentially of an amino acid sequence according to SEQ IDNO:1.

The present description further relates to the peptides according to thepresent description, wherein said peptide is modified and/or includesnon-peptide bonds.

The present description further relates to the peptides according to thepresent description, wherein said peptide is part of a fusion protein,in particular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present description further relates to a nucleic acid, encoding thepeptides according to the present description. The present descriptionfurther relates to the nucleic acid according to the present descriptionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present description further relates to an expression vector capableof expressing and/or expressing a nucleic acid according to the presentdescription.

The present description further relates to a peptide according to thepresent description, a nucleic acid according to the present descriptionor an expression vector according to the present description for use inthe treatment of diseases and in medicine, in particular in thetreatment of cancer.

The present description further relates to antibodies that are specificagainst the peptides according to the present description or complexesof said peptides according to the present description with MHC, andmethods of making these.

The present description further relates to a host cell comprising anucleic acid according to the present description or an expressionvector as described before.

The present description further relates to the host cell according tothe present description that is an antigen presenting cell, andpreferably is a dendritic cell.

The present description further relates to a method for producing apeptide according to the present description, said method comprisingculturing the host cell according to the present description, andisolating the peptide from said host cell or its culture medium.

The present description further relates to said method according to thepresent description, wherein the antigen is loaded onto class I or IIMHC molecules expressed on the surface of a suitable antigen-presentingcell or artificial antigen-presenting cell by contacting a sufficientamount of the antigen with an antigen-presenting cell.

The present description further relates to the method according to thepresent description, wherein the antigen-presenting cell comprises anexpression vector capable of expressing and/or expressing said peptidecontaining SEQ ID NO:1, preferably containing SEQ ID NO:1, or a variantamino acid sequence.

The present description further relates to activated T-cells, producedby the method according to the present description, wherein said T-cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present description.

The present description further relates to a method of killingtarget-cells in a patient which target-cells aberrantly express apolypeptide comprising any amino acid sequence according to the presentdescription, the method comprising administering to the patient aneffective number of T-cells as produced according to the presentdescription.

The present description further relates to the use of any peptide asdescribed, the nucleic acid according to the present description, theexpression vector according to the present description, the cellaccording to the present description, the activated T lymphocyte, theT-cell receptor or the antibody or other peptide- and/orpeptide-MHC-binding molecules according to the present description as amedicament or in the manufacture of a medicament. Preferably, saidmedicament is active against cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present description further relates to a use according to thepresent description, wherein said cancer cells are from gastric cancer,prostate carcinoma, oral cavity carcinomas, oral squamous carcinoma(OSCC), acute myeloid leukemia (AML), H. pylori-induced MALT lymphoma,colon carcinoma/colorectal cancer, glioblastoma, non-small-cell lungcancer (NSCLC), cervical carcinoma, human breast cancer, prostatecancer, colon cancer, pancreatic cancers, pancreatic ductaladenocarcinoma, ovarian cancer, hepatocellular carcinoma, liver cancer,brain tumors of different phenotypes, leukemias such as acutelymphoblastic leukemia (ALL), lung cancer, Ewing's sarcoma, endometrialcancer, head and neck squamous cell carcinoma, epithelial cancer of thelarynx, oesophageal carcinoma, oral carcinoma, carcinoma of the urinarybladder, ovarian carcinomas, renal cell carcinoma, atypical meningioma,papillary thyroid carcinoma, brain tumors, salivary duct carcinoma,cervical cancer, extranodal T/NK-cell lymphomas, Non-Hodgkins Lymphomaand malignant solid tumors of the lung and breast and other tumors.

The present description further relates to biomarkers based on thepeptides according to the present description, herein called “targets,”that can be used in the diagnosis of cancer, preferably non-small celllung cancer. The marker can be over-presentation of the peptide(s)themselves, or over-expression of the corresponding gene(s). The markersmay also be used to predict the probability of success of a treatment,preferably an immunotherapy, and most preferred an immunotherapytargeting the same target that is identified by the biomarker. Forexample, an antibody or soluble TCR can be used to stain sections of thetumor to detect the presence of a peptide of interest in complex withMHC. Optionally the antibody carries a further effector function such asan immune stimulating domain or toxin.

The present description further relates to the use of these noveltargets for the identification of TCRs that recognize at least one ofsaid targets, and preferably the identification of said TCRs thatactivate T-cells.

The present description also relates to the use of these novel targetsin the context of cancer treatment.

The present description further relates to the use of the peptidesaccording to the invention for the production of TCRs, individual TCRsubunits (alone or in combination), and subdomains thereof, inparticular soluble TCR (sTCRs) and cloned TCRs, said TCRs engineeredinto autologous or allogeneic T-cells, and methods of making same, aswell as other cells bearing said TCR or cross-reacting with said TCRs.

The present description further relates to a TCR protein, individual TCRsubunits (alone or in combination), and subdomains thereof, inparticular soluble TCR (sTCRs) and cloned TCRs that bind to a KIQEILTQV(SEQ ID NO:1)-HLA-A*02 complex comprising a TCR alpha chain variabledomain and a TCR beta chain variable domain.

The present description further relates to an isolated nucleic acidcomprising a nucleotide sequence encoding a TCR of the presentdescription. The present description further relates to a recombinantexpression vector comprising a nucleic acid encoding a TCR alpha chain,beta chain, or both, as produced according to the present description.

The present description further relates to an isolated host cellcomprising the recombinant expression vector expressing the nucleic acidencoding the TCR alpha chain, beta chain, or both, of the presentdescription.

The present description further relates to an isolated host cellcomprising the recombinant expression vector of the present description,preferably wherein the cell is a peripheral blood lymphocyte (PBL).

The present description further relates to an isolated PBL comprisingthe recombinant expression vector of the present description, whereinthe PBL is a CD8+ T-cell or a CD4+ T-cell.

The present description further relates to a population of cellscomprising at least one host cell of the present description.

The present description further relates to TCR proteins of the presentdescription for use in the treatment of proliferative diseases, such as,non-small cell lung cancer, small cell lung cancer, renal cell cancer,brain cancer, gastric cancer, colorectal cancer, hepatocellular cancer,pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma, melanoma, ovarian cancer, urinary bladder cancer, uterinecancer, gallbladder and bile duct cancer and esophageal cancer.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T-cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of thedescription) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present description differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the description, as longas the correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the description as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentdescription), if it is capable of inducing an immune response. In thecase of the present description, immunogenicity is more specificallydefined as the ability to induce a T-cell response. Thus, an “immunogen”would be a molecule that is capable of inducing an immune response, andin the case of the present description, a molecule capable of inducing aT-cell response. In another aspect, the immunogen can be the peptide,the complex of the peptide with MHC, oligopeptide, and/or protein thatis used to raise specific antibodies or TCRs against it.

A class I T-cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by aT-cell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

Table 1: Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F=1−(1−Gf)².Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004).

Calculated phenotype Allele Population from allele frequency A*02Caucasian (North America)  49.1% A*02 African American (North America) 34.1% A*02 Asian American (North America)  43.2% A*02 Latin American(North American)  48.3% DR1 Caucasian (North America)  19.4% DR2Caucasian (North America)  28.2% DR3 Caucasian (North America)  20.6%DR4 Caucasian (North America)  30.7% DR5 Caucasian (North America) 23.3% DR6 Caucasian (North America)  26.7% DR7 Caucasian (NorthAmerica)  24.8% DR8 Caucasian (North America)  5.7% DR9 Caucasian (NorthAmerica)  2.1% DR1 African (North) American 13.20% DR2 African (North)American 29.80% DR3 African (North) American 24.80% DR4 African (North)American 11.10% DR5 African (North) American 31.10% DR6 African (North)American 33.70% DR7 African (North) American 19.20% DR8 African (North)American 12.10% DR9 African (North) American  5.80% DR1 Asian (North)American  6.80% DR2 Asian (North) American 33.80% DR3 Asian (North)American  9.20% DR4 Asian (North) American 28.60% DR5 Asian (North)American 30.00% DR6 Asian (North) American 25.10% DR7 Asian (North)American 13.40% DR8 Asian (North) American 12.70% DR9 Asian (North)American 18.60% DR1 Latin (North) American 15.30% DR2 Latin (North)American 21.20% DR3 Latin (North) American 15.20% DR4 Latin (North)American 36.80% DR5 Latin (North) American 20.00% DR6 Latin (North)American 31.10% DR7 Latin (North) American 20.20% DR8 Latin (North)American 18.60% DR9 Latin (North) American  2.10% A*24 Philippines   65%A*24 Russia Nenets   61% A*24:02 Japan   59% A*24 Malaysia   58% A*24:02Philippines   54% A*24 India   47% A*24 South Korea   40% A*24 Sri Lanka  37% A*24 China   32% A*24:02 India   29% A*24 Australia West   22%A*24 USA   22% A*24 Russia Samara   20% A*24 South America   20% A*24Europe   18%

In an embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this description are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein the term “a nucleotide coding for (or encoding) a TCRprotein” refers to a nucleotide sequence coding for the TCR proteinincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example,T-cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. In an aspect,such polynucleotides are part of a vector and/or such polynucleotides orpolypeptides are part of a composition, and still be isolated in thatsuch vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present description may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present description, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present description can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present description, the term “percent identity”or “percent identical”, when referring to a sequence, means that asequence is compared to a claimed or described sequence after alignmentof the sequence to be compared (the “Compared Sequence”) with thedescribed or claimed sequence (the “Reference Sequence”). The percentidentity is then determined according to the following formula:percent identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iv) the alignment has to start at position 1 of the aligned sequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present description thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO:1 to SEQ ID NO:10 or a variant thereof which is 85% homologousto SEQ ID NO:1 to SEQ ID NO:10, or a variant thereof that will induceT-cells cross-reacting with said peptide. The peptides of thedescription have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present description, the term “homologous” refers to the degreeof identity (see percent identity above) between sequences of two aminoacid sequences, i.e., peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T-cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence consisting of SEQ ID NO:1. Preferred is KIQEILTQV (SEQ IDNO:1). For example, a peptide may be modified so that it at leastmaintains, if not improves, the ability to interact with and bind to thebinding groove of a suitable MHC molecule, such as HLA-A*02 or -DR, andin that way it at least maintains, if not improves, the ability to bindto the TCR of activated T-cells. Similarly, a TCR protein may bemodified so that it at least maintains, if not improves, the ability tointeract with and bind to a suitable MHC molecule/KIQEILTQV (SEQ IDNO:1) complex, such as HLA-A*02 or -DR, and in that way it at leastmaintains, if not improves, the ability to activate T-cells.

These T-cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide, such as KIQEILTQV (SEQ ID NO:1), as defined inthe aspects of the description. As can be derived from the scientificliterature and databases (Rammensee et al., 1999; Godkin et al., 1997),certain positions of HLA binding peptides are typically anchor residuesforming a core sequence fitting to the binding motif of the HLAreceptor, which is defined by polar, electrophysical, hydrophobic andspatial properties of the polypeptide chains constituting the bindinggroove. Thus, one skilled in the art would be able to modify the aminoacid sequence set forth in SEQ ID NO:1, by maintaining the known anchorresidues, and would be able to determine whether such variants maintainthe ability to bind MHC class I or II molecules/KIQEILTQV (SEQ ID NO:1)complexes. The variants of the present description retain the ability tobind MHC class I or II molecules/KIQEILTQV (SEQ ID NO:1) complexes.T-cells expressing the variants of the present description cansubsequently kill cells that express a polypeptide containing thenatural amino acid sequence of the cognate peptide, such as KIQEILTQV(SEQ ID NO:1).

The original (unmodified) peptides or TCR proteins as disclosed hereincan be modified by the substitution of one or more residues atdifferent, possibly selective, sites within the peptide chain, if nototherwise stated. Preferably those substitutions are located at the endof the amino acid chain of said peptide. For TCR proteins, preferablythose substitutions are located at variable domains of TCR alpha chainand TCR beta chain. Such substitutions may be of a conservative nature,for example, where one amino acid is replaced by an amino acid ofsimilar structure and characteristics, such as where a hydrophobic aminoacid is replaced by another hydrophobic amino acid. Even moreconservative would be replacement of amino acids of the same or similarsize and chemical nature, such as where leucine is replaced byisoleucine. In studies of sequence variations in families of naturallyoccurring homologous proteins, certain amino acid substitutions are moreoften tolerated than others, and these are often show correlation withsimilarities in size, charge, polarity, and hydrophobicity between theoriginal amino acid and its replacement, and such is the basis fordefining “conservative substitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of thedescription and yet still be encompassed by the disclosure herein.

In addition, non-standard amino acids (i.e., other than the commonnaturally occurring proteinogenic amino acids) may also be used forsubstitution purposes to produce immunogens and immunogenic polypeptidesaccording to the present description.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the TCR can be modified by replacement with otheramino acids whose incorporation do not substantially affect T-cellreactivity and does not eliminate binding to the relevant MHC. Thus,apart from the proviso given, the peptide of the description may be anypeptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 2 Variants of the peptide of the invention Position: 1 2 3 4 5 6 78 9 IGF2BP3-001 (SEQ ID K I Q E I L T Q V NO: 1) IGF2BP3-001 A1 (SEQ IDA I Q E I L T Q V NO: 2) IGF2BP3-001 A2 (SEQ ID K A Q E I L T Q V NO: 3)IGF2BP3-001 A3 (SEQ ID K I A E I L T Q V NO: 4) IGF2BP3-001 A4 (SEQ ID KI Q A I L T Q V NO: 5) IGF2BP3-001 A5 (SEQ ID K I Q E A L T Q V NO: 6)IGF2BP3-001 A6 (SEQ ID K I Q E I A T Q V NO: 7) IGF2BP3-001 A7 (SEQ ID KI Q E I L A Q V NO: 8) IGF2BP3-001 A8 (SEQ ID K I Q E I L T A V NO: 9)IGF2BP3-001 A9 (SEQ ID K I Q E I L T Q A NO: 10)

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually the actual epitope are residuesthat do not substantially affect proteolytic cleavage necessary toexpose the actual epitope during processing.

The peptides of the description can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 8 and 11 amino acids long, are generated bypeptide processing from longer peptides or proteins that include theactual epitope. It is preferred that the residues that flank between 4:0and 0:4. Combinations of the elongations according to the descriptioncan be found in Table 3.

TABLE 3 Combinations of the elongations of peptides of the descriptionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present description may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present description provides peptides and variants ofMHC class I epitopes, wherein the peptide or variant has an overalllength of between 8 and 100, preferably between 8 and 30, and mostpreferred between 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids,in case of the elongated class II binding peptides the length can alsobe 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present descriptionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T-cells specific for a peptide according to thepresent description are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T-cells from more than oneindividual, at least two, and more preferably three individuals.

Affinity-enhancement of tumor-specific TCRs, and its exploitation,relies on the existence of a window for optimal TCR affinities. Theexistence of such a window is based on observations that TCRs specificfor HLA-A2-restricted pathogens have KD values that are generally about10-fold lower when compared to TCRs specific for HLA-A2-restrictedtumor-associated self-antigens (Aleksic et al. 2012; Kunert et al.2013). It is now known, although tumor antigens have the potential to beimmunogenic, because tumors arise from the individual's own cells onlymutated proteins or proteins with altered translational processing willbe seen as foreign by the immune system. Antigens that are upregulatedor overexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance (Xinget al. 2012; Ruella et al. 2014; Sharpe et al. 2015), meaning that onlyT-cells with low-affinity TCRs for self antigens remain. Therefore,affinity of TCRs or variants of the present description to MAG-003 havebeen enhanced by methods well known in the art as described below.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides or TCR proteinseither in the free form or in the form of a pharmaceutically acceptablesalt (see also above). As used herein, “a pharmaceutically acceptablesalt” refers to a derivative of the disclosed peptides wherein thepeptide is modified by making acid or base salts of the agent. Forexample, acid salts are prepared from the free base (typically whereinthe neutral form of the drug has a neutral —NH2 group) involvingreaction with a suitable acid. Suitable acids for preparing acid saltsinclude both organic acids, e.g., acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinicacid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoicacid, cinnamic acid, mandelic acid, methane sulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, aswell as inorganic acids, e.g., hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid phosphoric acid and the like.

Conversely, preparation of basic salts of acid moieties which may bepresent on a peptide are prepared using a pharmaceutically acceptablebase such as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine or the like.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 10 andhas been synthetically produced (e.g. synthesized) as a pharmaceuticallyacceptable salt. Methods to synthetically produce peptides are wellknown in the art. The salts of the peptides according to the presentinvention differ substantially from the peptides in their state(s) invivo, as the peptides as generated in vivo are no salts. The non-naturalsalt form of the peptide mediates the solubility of the peptide, inparticular in the context of pharmaceutical compositions comprising thepeptides, e.g. the peptide vaccines as disclosed herein. A sufficientand at least substantial solubility of the peptide(s) is required inorder to efficiently provide the peptides to the subject to be treated.Preferably, the salts are pharmaceutically acceptable salts of thepeptides. These salts according to the invention include alkaline andearth alkaline salts such as salts of the Hofmeister series comprisingas anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, SCN⁻ andas cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄, (NH₄)₂HPO₄,(NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃, NH₄ClO₄, NH₄I,NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br,Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄, K₂SO₄, KCH₃COO,KCl, KBr, KNO₃, KClO₄, Kl, KSCN, Na₃PO₄, Na₂HPO₄, NaH₂PO₄, Na₂SO₄,NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂ Cs₃PO₄, Cs₂HPO₄,CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄, CsI, CsSCN,Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr, LiNO₃, LiClO₄,LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂,MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄),Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂, Ca(NO₃)₂,Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄,Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, Bale, and Ba(SCN)₂.Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCl, NaCl,and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides or TCR proteins as salts of acetic acid(acetates), trifluoro acetates or hydrochloric acid (chlorides).

A further aspect of the description provides a nucleic acid (for examplea polynucleotide) encoding a peptide or peptide variant and a TCRprotein and TCR variants of the description. The polynucleotide may be,for example, DNA, cDNA, PNA, RNA or combinations thereof, either single-and/or double-stranded, or native or stabilized forms ofpolynucleotides, such as, for example, polynucleotides with aphosphorothioate backbone and it may or may not contain introns so longas it codes for the peptide. Of course, only peptides that containnaturally occurring amino acid residues joined by naturally occurringpeptide bonds are encodable by a polynucleotide. A still further aspectof the description provides an expression vector capable of expressing apolypeptide according to the description.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of thedescription employs the polymerase chain reaction as disclosed by SaikiR K, et al. (Saiki et al., 1988). This method may be used forintroducing the DNA into a suitable vector, for example by engineeringin suitable restriction sites, or it may be used to modify the DNA inother useful ways as is known in the art. If viral vectors are used,pox- or adenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the description. Thus, the DNA encoding thepeptide or variant of the description may be used in accordance withknown techniques, appropriately modified in view of the teachingscontained herein, to construct an expression vector, which is then usedto transform an appropriate host cell for the expression and productionof the polypeptide of the description. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the description may be joinedto a wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of thedescription are then cultured for a sufficient time and underappropriate conditions known to those skilled in the art in view of theteachings disclosed herein to permit the expression of the polypeptide,which can then be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.),planT-cells, animal cells and insecT-cells. Preferably, the system canbe mammalian cells such as CHO cells available from the ATCC CellBiology Collection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potenT-cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of thedescription are encoded and thus expressed in a successive order(similar to “beads on a string” constructs). In doing so, the peptidesor peptide variants may be linked or fused together by stretches oflinker amino acids, such as for example LLLLLL, or may be linked withoutany additional peptide(s) between them. These constructs can also beused for cancer therapy, and may induce immune responses both involvingMHC I and MHC II.

The present description also relates to a host cell transformed with apolynucleotide vector construct of the present description. The hostcell can be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insecT-cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent description is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeasT-cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeasT-cell,bacterial cells, insecT-cells and vertebrate cells.

Successfully transformed cells, i.e., cells that contain a DNA constructof the present description, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the description areuseful in the preparation of the peptides of the description, forexample bacterial, yeast and insecT-cells. However, other host cells maybe useful in certain therapeutic methods. For example,antigen-presenting cells, such as dendritic cells, may usefully be usedto express the peptides of the description such that they may be loadedinto appropriate MHC molecules. Thus, the current description provides ahost cell comprising a nucleic acid or an expression vector according tothe description.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the description provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the TCR proteins, the nucleic acid or theexpression vector of the description are used in medicine. For example,the peptide or its variant may be prepared for intravenous (i.v.)injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g., between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g., Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T-cells for the respectiveopposite CDR as noted above.

The medicament of the description may also include one or moreadjuvants. Adjuvants are substances that non-specifically enhance orpotentiate the immune response (e.g., immune responses mediated byCD8-positive T-cells and helper-T (TH) cells to an antigen, and wouldthus be considered useful in the medicament of the present description.Suitable adjuvants include, but are not limited to, 1018 ISS, aluminumsalts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellinor TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31,Imiquimod (ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2,IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivativesthereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2,MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206,Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-wateremulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vectorsystem, poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T-cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present description. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g., CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g., AmpliGen®, Hiltonal®,poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA aswell as immunoactive small molecules and antibodies such ascyclophosphamide, sunitinib, Bevacizumab®, celebrex, NCX-4016,sildenafil, tadalafil, vardenafil, sorafenib, temozolomide,temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171,anti-CTLA4, other antibodies targeting key structures of the immunesystem (e.g., anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives useful in thecontext of the present description can readily be determined by theskilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe description the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe description the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the description, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonal®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the description attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g., antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g., a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g., the complex of a peptide with MHC,according to the application at hand). In another embodiment a scaffoldis able to activate signaling through its target antigen, for example aT-cell receptor complex antigen. Scaffolds include but are not limitedto antibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T-cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e., not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labeling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labeledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present description further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. In anaspect, at least one or more aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides according to the description athand with the MHC molecule, using the cell-SELEX (Systematic Evolutionof Ligands by Exponential enrichment) technique.

The peptides of the present description can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the description to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the description to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present description are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present description.

The present description relates to a TCR protein or a variant orfunctional fragment thereof that specifically binds to SEQ ID NO:1.

The present description further relates to the TCR protein according tothe description, wherein the TCR protein is (chemically) modified and/orincludes non-peptide bonds.

The present description further relates to a nucleic acid, encoding theTCR proteins according to the description, provided that the TCR proteinis not the complete (full) human protein.

The present description further relates to the nucleic acid according tothe description that is DNA, cDNA, PNA, RNA or combinations thereof.

The present description further relates to an expression vector capableof expressing a nucleic acid according to the present description.

The present description further relates to a TCR protein according tothe present description, a nucleic acid according to the presentdescription or an expression vector according to the present descriptionfor use in medicine, in particular in the (improved) treatment ofgastric cancer, colorectal cancer and/or glioblastoma.

The present description further relates to a host cell comprising anucleic acid according to the description or an expression vectoraccording to the description.

The present description further relates to the host cell according tothe present description that is a T-cell, and preferably a CD8-positiveT-cell or CD4-positive T-cell.

The present description further relates to a method of producing a TCRprotein according to the present description, said method comprisingincubating PBMCs from HLA-A*02-negative healthy donors withA2/IGF2BP3-001 (SEQ ID NO:1) monomers, incubating the PBMCs withtetramer-phycoerythrin (PE) and isolating the high avidity T-cells byfluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of producing a TCRprotein according to the present description, said method comprisingincubating PBMCs from HLA-A*02-negative healthy donors with A2/p286-1Y2Lmonomers, incubating the PBMCs with tetramer-phycoerythrin (PE) andisolating the high avidity T-cells by fluorescence activated cellsorting (FACS)-Calibur analysis.

The present description further relates to a method of producing a TCRprotein according to the present description, said method comprisingincubating PBMCs from HLA-A*02-negative healthy donors withA2/p286-1Y2L9L monomers, incubating the PBMCs withtetramer-phycoerythrin (PE) and isolating the high avidity T-cells byfluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of producing a TCRprotein according to the present description, said method comprisingobtaining a transgenic mouse with the entire human TCRαβ gene loci (1.1and 0.7 Mb), whose T-cells express a diverse human TCR repertoire thatcompensates for mouse TCR deficiency, immunizing the mouse withIGF2BP3-001, incubating PBMCs obtained from the transgenic mice withtetramer-phycoerythrin (PE), and isolating the high avidity T-cells byfluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of producing a TCRprotein according to the present description, said method comprisingobtaining a transgenic mouse with the entire human TCRαβ gene loci (1.1and 0.7 Mb), whose T-cells express a diverse human TCR repertoire thatcompensates for mouse TCR deficiency, immunizing the mouse withp286-1Y2L, incubating PBMCs obtained from the transgenic mice withtetramer-phycoerythrin (PE), and isolating the high avidity T-cells byfluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of producing a TCRprotein according to the present description, said method comprisingobtaining a transgenic mouse with the entire human TCRαβ gene loci (1.1and 0.7 Mb), whose T-cells express a diverse human TCR repertoire thatcompensates for mouse TCR deficiency, immunizing the mouse withp286-1Y2L9L, incubating PBMCs obtained from the transgenic mice withtetramer-phycoerythrin (PE), and isolating the high avidity T-cells byfluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of killing targetcells in a patient which target cells aberrantly express IGF2BP3-001,the method comprising administering to the patient an effective numberof T-cells as according to the present description.

The present description further relates to the use of any TCR proteindescribed, a nucleic acid according to the present description, anexpression vector according to the present description, a cell accordingto the present description, or an activated cytotoxic T lymphocyteaccording to the present description as a medicament or in themanufacture of a medicament. The present description further relates toa use according to the present description, wherein the medicament isactive against cancer.

The present description further relates to a use according to thedescription, wherein said cancer cell is selected from gastric cancer,prostate carcinoma, oral cavity carcinomas, oral squamous carcinoma(OSCC), acute myeloid leukemia (AML), H. pylori-induced MALT lymphoma,colon carcinoma/colorectal cancer, glioblastoma, non-small-cell lungcancer (NSCLC), cervical carcinoma, human breast cancer, prostatecancer, colon cancer, pancreatic cancers, pancreatic ductaladenocarcinoma, ovarian cancer, hepatocellular carcinoma, liver cancer,brain tumors of different phenotypes, leukemias such as acutelymphoblastic leukemia (ALL), lung cancer, Ewing's sarcoma, endometrialcancer, head and neck squamous cell carcinoma, epithelial cancer of thelarynx, oesophageal carcinoma, oral carcinoma, carcinoma of the urinarybladder, ovarian carcinomas, renal cell carcinoma, atypical meningioma,papillary thyroid carcinoma, brain tumors, salivary duct carcinoma,cervical cancer, extranodal T/NK-cell lymphomas, Non-Hodgkins Lymphomaand malignant solid tumors of the lung and breast and other tumors.

The present description further relates to particular marker proteinsand biomarkers based on the peptides according to the presentdescription, herein called “targets” that can be used in the diagnosisand/or prognosis of the camcers as above, in particular gastric cancer,colorectal cancer and glioblastoma. The present description also relatesto the preferred use of these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g., CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a gastric cancer, colorectalcancer and glioblastoma marker (poly)peptide, delivery of a toxin to agastric cancer, colorectal cancer and glioblastoma cancer cellexpressing a cancer marker gene at an increased level, and/or inhibitingthe activity of a gastric cancer, colorectal cancer and glioblastomacancer marker polypeptide) according to the description.

Whenever possible, the antibodies of the description may be purchasedfrom commercial sources. The antibodies of the description may also begenerated using well-known methods. The skilled artisan will understandthat either full length non-small cell lung cancer marker polypeptidesor fragments thereof may be used to generate the antibodies of thedescription. A polypeptide to be used for generating an antibody of thedescription may be partially or fully purified from a natural source, ormay be produced using recombinant DNA techniques.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the description may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the description can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the description may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the description are preferably administered to a subjectin a pharmaceutically acceptable carrier. Typically, an appropriateamount of a pharmaceutically-acceptable salt is used in the formulationto render the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating non-small celllung cancer, the efficacy of the therapeutic antibody can be assessed invarious ways well known to the skilled practitioner. For instance, thesize, number, and/or distribution of cancer in a subject receivingtreatment may be monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the description to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g., by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. In another aspect, it is expressedin T-cells used for adoptive transfer. See, for example, WO2004/033685A1, WO 2004/074322A1, and WO 2013/057586A1, the contents ofwhich are incorporated by reference in their entirety.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present description can be used to verify apathologist's diagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

The present invention will be further described in the followingexamples, nevertheless, without being limited thereto. For the purposesof the present invention, all references as cited herein areincorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows IGF2BP3-001 peptide presentation in normal tissues andcancers.

FIG. 2 shows IGF2BP3-001 expression in cancer and normal tissues.

FIG. 3 shows IGF2BP3-001 expression in cancer and normal tissues.

FIG. 4 shows IGF2BP3-001 expression in cancer and normal tissues.

FIG. 5 shows affinities of IGF2BP3 to HLA-A*02:01 Dissociation constants(KD) of IGF2BP3 and control peptides MUC-001 (intermediate binder) andMET-001 (strong binder) were measured by an ELISA-based assay.

FIG. 6 shows that IGF2BP3 mRNA is detectable in all evaluated cancerspecimens as in Example 6. The level of expression covers a range fromconsiderable expression in colorectal cancer, head and neck cancer,non-small cell lung cancer, ovarian cancer and esophageal cancerspecimens (CCA001T, CCA006T, HNSCC017T1, NSCLCOO4T1, NSCLCOO5T1, 00038T,OSCAR052T1 and OSCAR055T1) to rather low expression in a pancreaticcancer specimen (PC002T).

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES

HLA-Binding

The SYFPEITHI routine (Rammensee et al., 1997; Rammensee et al., 1999)predicts binding of KIQEILTQV (SEQ ID NO: 1) to A*02:01 with an absolutescore of 27 and a relative score of 0.74. The peptide IGF2BP3-001 waspresented on cancer cells as follows:

TABLE 4 IGF2BP3-001 CRC GC HCC NSCLC OC OSCAR PC RCC Naturalpresentation of IGF2BP3-001 peptide target (MS data) Naturalpresentation directly shown on tumor samples + + + + + + + +Presentation on normal tissues Not detected on normal tissues (n = 241samples) mRNA expression of IGF2BP3 source protein Over-expressed ontumor samples (% of analyzed 10 30 10 45 25 55 20 5 tumor samples)Over-expression reported in the literature + + + + + + + + High andmedium risk class normal tissues with High risk: Lung (0.09/0.21)highest expression (RPKM values median/Q95) Medium risk: Esophagus(0.47/1.85) Low risk: Testis (1.89/3.2) Immunogenicity and/or functionalT-cell data Immunogenic in a large fraction of tested A*02 positivedonors (69%). Primed T-cells are able to kill peptide- pulsed targetcells.

TABLE 5 Frequency of IGF2BP3-001 presentation A*02 Samples Meanintensity Normal  1 of 225 — Cancer 63 of 376 7.5e+06 cIPC 9 of 201.9e+07 CRC 3 of 28 1.0e+07 HCC 7 of 16 1.2e+07 OC 7 of 20 6.4e+06 OSCAR2 of 16 6.1e+06 PC 3 of 19 8.3e+06 pCLL 2 of 11 9.8e+06 pCRC 3 of 241.0e+07 pGB 5 of 28 5.6e+06 pGC 11 of 45  6.4e+06 pNSCLC 18 of 91 6.8e+06 pPC 3 of 18 8.3e+06 pRCC 3 of 18 1.4e+07 RCC 3 of 22 1.4e+07SCLC 2 of 13 6.46+06

IGF2BP3-001 was quantified in >380 HLA-A*02 positive tumor samplesincluding 16 different cancer types and >240 normal tissue samples ofdifferent origin covering all risk categories with a focus on high andmedium risk organs (status: August 2015). FIG. 1 shows the relativepeptide presentation levels of IGF2BP3-001 in these tissues. IGF2BP3-001was frequently and exclusively found on primary tumor samples ofdifferent entities and not on normal tissues. Thus, the IGF2BP3-001target is presented in the context of HLA-A*02 in a highlytumor-specific manner.

The data shown in FIG. 5 further provides evidence that IGF2BP3-001 is apeptide with very good binding to HLA-A*02:01.

Allo-reactive settings can be used to circumvent self-tolerance andyield T-cells with a higher avidity when compared to T-cells derivedfrom autologous settings, i.e., patients. Examples of such settingsinclude in vitro generation of allo-HLA reactive, peptide-specificT-cells (Sadovnikova et al. 1998; Savage et al. 2004; Wilde et al.2012), and immunization of mice transgenic for human-MHC or human TCR(Stanislawski et al. 2001; Li et al. 2010).

Example 1

In vitro generation of allo-HLA reactive, peptide-specific T-cells(Savage et al. 2004) PBMCs from HLA-A*02-positive and HLA-A*02-negativehealthy donors were used after obtaining informed consent. Recombinantbiotinylated HLA-A2 class I monomers and A2 fluorescent tetramerscontaining IGF2BP3-001 were obtained from MBLI (Woburn, Mass.). PBMCswere incubated with anti-CD20SA diluted in phosphate buffered saline(PBS) for 1 hour at room temperature, washed, and incubated with thebiotinylated A2/IGF2BP3-001 monomers for 30 minutes at room temperature,washed, and plated at 3×10⁶ cells/well in 24-well plates in RPMI with10% human AB serum. Interleukin 7 (IL-7; R&D Systems, Minneapolis,Minn.) was added on day 1 at 10 ng/mL and IL-2 (Chiron, Harefield,United Kingdom) was added at 10 U/mL on day 4. Over a 5-week periodcells were restimulated weekly with fresh PBMCs, mixed with respondercells at a 1:1 ratio, and plated at 3×10⁶/well in 24-well plates.

To obtain high avidity T-cells, approximately 10⁶ PBMCs withHLA-A2/IGF2BP3-001 tetramer-phycoerythrin (PE) (obtained from MBLI) wereincubated for 30 minutes at 37° C., followed by anti-CD8-fluoresceinisothiocyanate (FITC)/allophycocyanin (APC) for 20 minutes at 4° C.,followed by fluorescence activated cell sorting (FACS)-Calibur analysis.Sorting was done with a FACS-Vantage (Becton Dickinson, Cowley, Oxford,United Kingdom). Sorted tetramer-positive cells were expanded in 24-wellplates using, per well, 2×10⁵ sorted cells, 2×10⁶ irradiated A2-negativePBMCs as feeders, 2×10⁴ CD3/CD28 beads/mL (Dynal, Oslo Norway), and IL-2(1000 U/mL). The high avidity T-cells, thus obtained, were then used toidentify and isolate TCRs using techniques known in the art, such assingle cell 5′ RACE (Rapid Amplification of cDNA Ends). Non-redundantTCR DNAs were then analyzed for amino acid/DNA sequences determinationand cloning into expression vectors using methods well known in the art.

Example 2: Cloning of TCRs

Methods of cloning TCRs are known in the art, for example, as describedin U.S. Pat. No. 8,519,100, which is hereby incorporated by reference inits entirety for said methods. The alpha chain variable region sequencespecific oligonucleotide A1 (ggaattccatatgagtcaacaaggagaagaagatcc SEQ IDNO:11) which encodes the restriction site NdeI, an introduced methioninefor efficient initiation of expression in bacteria, and an alpha chainconstant region sequence specific oligonucleotide A2(ttgtcagtcgacttagagtctctcagctggtacacg SEQ ID NO:12) which encodes therestriction site SalI are used to amplify the alpha chain variableregion. In the case of the beta chain, a beta chain variable regionsequence specific oligonucleotide B1(tctctcatatggatggtggaattactcaatccccaa SEQ ID NO:13) which encodes therestriction site NdeI, an introduced methionine for efficient initiationof expression in bacteria, and a beta chain constant region sequencespecific oligonucleotide B2 (tagaaaccggtggccaggcacaccagtgtggc SEQ IDNO:14) which encodes the restriction site AgeI are used to amplify thebeta chain variable region.

Three TCRs (R10P1A7, R13P1C6, and R18P1C12), each encoding tumorspecific TCR-alpha and TCR-beta chains, were isolated and cloned fromT-cells of three healthy donors. TCR R18P1C12 was derived from HLA-A2positive donor and TCR R10P1A7 and TCR R13P1C6 were derived from HLA-A2negative donors.

The alpha and beta variable regions of the TCRs were sequenced. The TCRalpha and beta variable regions were then cloned into pGMT7-basedexpression plasmids containing either Cα or Cβ (respectively) bystandard methods described in (Molecular Cloning a Laboratory ManualThird edition by Sambrook and Russell). Plasmids were sequenced using anApplied Biosystems 3730×1 DNA Analyzer.

The DNA sequences encoding the TCR alpha chain cut with NdeI and SalIwere ligated into pGMT7+Cα vector, which was cut with NdeI and XhoI. TheDNA sequences encoding the TCR beta chain cut with NdeI and AgeI wasligated into separate pGMT7+Cβ vector, which was also cut with NdeI andAgeI. Ligated plasmids are transformed into competent Escherichia colistrain XL1-blue cells and plated out on LB/agar plates containing 100μg/ml ampicillin. Following incubation overnight at 37° C., singlecolonies are picked and grown in 10 ml LB containing 100 μg/mlampicillin overnight at 37° C. with shaking. Cloned plasmids arepurified using a Miniprep kit (Qiagen) and the insert is sequenced usingan automated DNA sequencer (Lark Technologies).

Phage display can be used to generate libraries of TCR variants toidentify high affinity mutants. The TCR phage display and screeningmethods described in (Li et al, (2005) Nature Biotech 23 (3): 349-354)can be applied to a reference TCR.

For example, all three CDR regions of the alpha chain sequence and allthree CDR regions of the beta chain sequence can be targeted bymutagenesis, and each CDR library panned and screened separately.

Accordingly, TCRs with affinities and/or binding half-lives at leasttwice that of the reference TCR (and therefore impliedly at least twicethat of the native TCR) can be identified.

TCR heterodimers are refolded using the method including the introducedcysteines in the constant regions to provide the artificial inter-chaindisulphide bond. In that way TCRs are prepared, consisting of (a) thereference TCR beta chain, together with mutated alpha chains; (b) thereference TCR alpha chain together with mutated beta chains; and (c)various combinations of beta and alpha chains including the mutantvariable domains.

The interaction between high affinity soluble disulfide-linked TCRs, andTCR variants, and the native peptide KIQEILTQV (SEQ ID NO: 1) HLA-A*02complex can be analyzed using the BIAcore method.

High avidity TCR variants can also be selected from a library of CDRmutants by yeast, or T-cell display (Holler et al. 2003; Chervin et al.2008). Candidate TCR variants, thus, provide guidance to designmutations of the TCR's CDRs to obtain high avidity TCR variants (Robbinset al. 2008; Zoete et al. 2007).

Example 3: Autologous T-Cell Engineering

T-cells can be engineered to express high avidity TCRs (so-called TCRtherapies) or protein-fusion derived chimeric antigen receptors (CARs)that have enhanced antigen specificity to MHC I/IGF2BP3-001 complex orMHC II/IGF2BP3-001 complex. In an aspect, this approach overcomes someof the limitations associated with central and peripheral tolerance, andgenerate T-cells that will be more efficient at targeting tumors withoutthe requirement for de novo T-cell activation in the patient.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding the tumor specific TCR-alpha and/orTCR-beta chains identified and isolated, as described in Examples 1-2,are cloned into expression vectors, such as gamma retrovirus orlentivirus. The recombinant viruses are generated and then tested forfunctionality, such as antigen specificity and functional avidity. Analiquot of the final product is then used to transduce the target T-cellpopulation (generally purified from patient PBMCs), which is expandedbefore infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs were synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAswere then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To test whether the exogenous TCRs were functionally expressed on cellsurface of the transformed T-cells, a tetramer staining technique wasused to detect MHC/IGF2BP3-001-binding T-cells. As shown in FIG. 5 andTable 6, a higher percentage of CD3-positive specific T-cell population,i.e., 6.78% (TCR R10P1A7) and 16.54% (TCR R13P1C6), was observed inTCR-expressing CD8+ T-cells by fluorescent-labeled MHC/IGF2BP3-001tetramer staining than that with MHC/unrelated peptide (e.g.,NYESO1-001) tetramers, e.g., 1.53%, or mock control, e.g., 1.73%. As acontrol, primary CD8+ T-cells transformed with unrelated TCRs, such as1G4 TCR, which is known to bind specifically to MHC/NYESO1-001 complex,was readily detected by MHC/NYESO1-001 tetramer, i.e., 17.69%. Theseresults indicate that TCR R10P1A7 and TCR R13P1C6 are expressed onT-cell surface and can bind specifically to MHC/IGF2BP3-001 complex.

To determine whether the TCRs induce MHC/IGF2BP3-001-specific cytotoxicactivity, the transformed CD8+ T-cells were co-incubated withIGF2BP3-001-loaded target cells or with target cells loaded with similarbut unrelated peptide, or with the controls, e.g., unloaded target cellsand CD8+ T-cells only, followed by IFN-γ release assay. IFN-γ secretionfrom CD8+ T-cells is indicative of T-cell activation with cytotoxicactivity.

TABLE 6 % specific TET in primary % TET Donor/HLA- IFNγ CD8+ IG4 TCRCode A2 (+ or −) (pg/ml) T-cells control EC50 R10P1A7 HBC-688/(−) 1756.78 1.53  ~1 nM R13P1C6 HBC-686/(−) 175-800 16.54 1.53 ~0.8 nM R18P1C12na/(+) 25-75 3.64 1.53 na

It was found that all primary CD8+ T-cells transformed with TCRs of thepresent disclosure, after co-incubation with IGF2BP3-001-loaded targetcells, released much higher levels of IFN-γ than that stimulated byunrelated peptide-loaded target cells, and the controls. Target peptidetitration analysis showed EC50 at ˜1 nM (TCR R10P1A7) and ˜0.8 nM (TCRR13P1C6). These results suggest that TCRs of the present invention canactivate cytotoxic T-cell activity, e.g., IFN-γ release, throughspecific interaction with the MHC/IGF2BP3-001 complex.

To determine the binding motif of the TCRs for the MHC/IGF2BP3-001complex, positional alanine scanning analysis was performed at each ofthe 9 amino acids of the IGF2BP3-001 peptide. Alanine-substitutedIGF2BP3-001 peptides are shown in Table 7.

TABLE 7 Position: 1 2 3 4 5 6 7 8 9 IGF2BP3-001 (SEQ ID K I Q E I L T QV NO: 1) IGF2BP3-001 A1 (SEQ ID A I Q E I L T Q V NO: 19) IGF2BP3-001 A2(SEQ ID K A Q E I L T Q V NO: 20) IGF2BP3-001 A3 (SEQ ID K I A E I L T QV NO: 21) IGF2BP3-001 A4 (SEQ ID K I Q A I L T Q V NO: 22) IGF2BP3-001A5 (SEQ ID K I Q E A L T Q V NO: 23) IGF2BP3-001 A6 (SEQ ID K I Q E I AT Q V NO: 24) IGF2BP3-001 A7 (SEQ ID K I Q E I L A Q V NO: 25)IGF2BP3-001 A8 (SEQ ID K I Q E I L T A V NO: 26) IGF2BP3-001 A9 (SEQ IDK I Q E I L T Q A NO: 27)

Briefly, T-cells transformed with each TCR were co-incubated with targetcells loaded with IGF2BP3-001, IGF2BP3-001-A1 to IGF2BP3-001-A9, or anunrelated NYESO1-001 peptide, followed by IFNγ release assay, asdescribed above.

Results of positional alanine scanning analysis on TCR R10P1A7 and TCRR13P1C6 are summarized in Table 8.

TABLE 8 TCR IGF2BP3-001 positions enable TCR binding R10P1A7 1, 3-7R13P1C6 3-6

A genome-wide screen for A*02-binding peptides with an identical motifrevealed no potentially cross-reactive peptides. These results suggestthat IGF2BP3-001 positions 3-6 may be important for TCR R10P1A7 and TCRR13P1C6 binding.

To determine efficacy of T-cells expressing TCRs described herein,primary CD8+ T-cells transformed with TCR R10P1A7 were co-incubated withhuman cancer cell lines, e.g., A-375 (human melanoma cell line) and T98G(human glioblastoma cell line), which are HLA-A2-positive andIGF2BP3-001 (target)-positive, and SK-BR-3 (human breast cancer cellline), which is HLA-A2-negative and IGF2BP3-001-negative, followed byIFNγ release assay.

IFNγ release was observed in both A-375 and T98G cells, which areHLA-A2-positive and IGF2BP3-001-positive, but not in SK-BR-3 cells,which have basal levels of IFNγ release that is comparable to that ofeffector cell only control. These results indicate that T-cellsexpressing TCR R10P1A7 can specifically induce cytotoxic activitytargeting cancer cells in a HLA-A2/IGF2BP3-001 specific manner.

The present description provides TCRs that are useful in treatingcancers/tumors, preferably melanoma and glioblastoma that over- orexclusively present IGF2BP3-001.

Example 4: Allogeneic T-Cell Engineering

Gamma delta (γδ) T cells, which are non-conventional T lymphocyteeffectors implicated in the first line of defense against pathogens, caninteract with and eradicate tumor cells in a MHC-independent mannerthrough activating receptors, among others, TCR-gamma and TCR-deltachains. These γδ T cells display a preactivated phenotype that allowsrapid cytokine production (IFN-γ, TNF-α) and strong cytotoxic responseupon activation. These T-cells have anti-tumor activity against manycancers and suggest that γδ T cell-mediated immunotherapy is feasibleand can induce objective tumor responses. (Braza et al. 2013).

Recent advances using immobilized antigens, agonistic monoclonalantibodies (mAbs), tumor-derived artificial antigen presenting cells(aAPC), or combinations of activating mAbs and aAPC have been successfulin expanding gamma delta T-cells with oligoclonal or polyclonal TCRrepertoires. For example, immobilized major histocompatibility complexClass-I chain-related A was a stimulus for γδ T-cells expressing TCRδ1isotypes, and plate-bound activating antibodies have expanded Vδ1 andVδ2 cells ex vivo. Clinically sufficient quantities of TCRδ1, TCRδ2, andTCRδ1^(neg)TCRδ2^(neg) have been produced following co-culture on aAPC,and these subsets displayed differences in memory phenotype andreactivity to tumors in vitro and in vivo. (Deniger et al. 2014).

In addition, γδ T-cells are amenable to genetic modification asevidenced by introduction of TCR-alpha and TCR-beta chains. (Hiasa etal. 2009). Another aspect of the present description relates toproduction of γδ T-cells expressing TCR-alpha and TCR-beta that bind toIGF2BP3-001. To do so, γδ T-cells are expanded by methods described byDeniger et al. 2014, followed by transducing the recombinant virusesexpressing the TCRs that bind to IGF2BP3-001 (as described in Example 3)into the expanded γδ T-cells. The virus-transduced γδ T-cells are theninfused into the patient.

Example 5: mRNA Expression

In situ hybridization (ISH) was used to detect mRNA expression directlyin formalin-fixed or frozen tissue sections. Due to its high sensitivityand its spatial resolution, it is a suitable method to determine celltype specific target expression and the distribution or frequency oftarget expression within cancer tissue sections.

ISH has been performed to detect IGF2BP3 mRNA using the RNAscope®technology developed by Advanced Cell Diagnostics (ACD). The RNAscope®technology is based on the hybridization of around 20 pairs of Z-shapedoligonucleotide probes to the target sequence. Signal amplification isachieved by branched DNA amplification, which is based on multiplehybridization steps of oligonucleotides, ultimately building up abranched DNA (bDNA) tree. Finally, a great number of label probeshybridize to the branches of the bDNA tree and the enhanced signal canbe detected. The chromogenic RNAscope® Detection Kit (RED) includeslabel probes which are linked to an enzyme (alkaline phosphatase).Signal detection depends on the enzymatic conversion of the chromogenicsubstrate FastRed, which additionally amplifies the original signal.RNAscope® is a very sensitive technology, which is due to the efficientprocess of signal amplification, paired with the high sensitivity andthe robust binding of the Z probe pairs to the target mRNA, even if itis partially crosslinked or degraded. According to ACD, binding of threeout of 20 probe pairs to each single RNA molecule is enough to generatea detectable ISH signal.

Each ISH experiment is subdivided into two methodological processes: 1)Tissue pretreatment for target retrieval, and 2) Target hybridization,signal amplification and detection. Optimal pretreatment conditions arecritical for successful target detection in FFPE tissue sections. Thefixation process induces crosslinking of proteins, DNA and RNA in cellsand tissues and thereby masks hybridization sites. Thus, to assureaccessibility of the target mRNA and proper binding of the probe set,these crosslinks have to be removed prior to target hybridization.Tissue pretreatment includes three discrete steps: 1) Blocking ofendogenous alkaline phosphatase by hydrogen peroxide treatment, 2)target retrieval by boiling in target retrieval reagent, and 3) targetretrieval by protease digestion. As the extent of fixation andcrosslinking may vary between different FFPE blocks, the optimal targetretrieval conditions have to be determined experimentally for eachindividual FFPE block. Therefore, tissue sections were exposed todifferent boiling and protease digestion times followed by hybridizationwith a positive and a negative control probe set. The optimal conditionswere determined by microscopic evaluation of specific signal intensityin the positive control, unspecific background in the negative controland tissue morphology. Tissue pretreatment was performed according tothe manufacturer's protocols. Pretreatment reagents are included in theRNAscope® reagent kits. After completion of the different pretreatmentsteps, target expression was assessed by hybridization of specific probesets to the mRNA of interest with subsequent branched DNA signalamplification and chromogenic or fluorescent signal detection. Allassays were performed according to the manufacturer's protocols.

TABLE 9 mRNA expression (see FIG. 6) Sample Tissue IGF2BP3 expressionCCA001T Colorectal cancer ++ CCA006T Colorectal cancer ++ HNSCC017T1Head and neck cancer ++ NSCLC004T1 Non-small cell lung cancer ++NSCLC005T1 Non-small cell lung cancer ++ OC038T1 Ovarian cancer ++OSCAR052T1 Esophageal cancer ++ OSCAR055T1 Esophageal cancer ++ PC002TPancreatic cancer + Overall expression level of IGF2BP3 in therespective section: ± very low, + low to moderate, ++ strong, +++ verystrong

REFERENCE LIST

-   Brossart P, Bevan M J. Presentation of exogenous protein antigens on    major histocompatibility complex class I molecules by dendritic    cells: pathway of presentation and regulation by cytokines. Blood.    1997 Aug. 15; 90(4):1594-9.-   Gnjatic S, Atanackovic D, Jäger E, Matsuo M, Selvakumar A, Altorki N    K, Maki R G, Dupont B, Ritter G, Chen Y T, Knuth A, Old L J. Survey    of naturally occurring CD4+ T cell responses against NY-ESO-1 in    cancer patients: correlation with antibody responses. Proc Natl Acad    Sci USA. 2003 Jul. 22; 100(15):8862-7.-   Mortara L, Castellani P, Meazza R, Tosi G, De Lerma Barbaro A,    Procopio F A, Comes A, Zardi L, Ferrini S, Accolla R S.    CIITA-induced MHC class II expression in mammary adenocarcinoma    leads to a Th1 polarization of the tumor microenvironment, tumor    rejection, and specific antitumor memory. Clin Cancer Res. 2006 Jun.    1; 12(11 Pt 1):3435-43.-   Dengjel J, Nastke M D, Gouttefangeas C, Gitsioudis G, Schoor O,    Altenberend F, Müller M, Krämer B, Missiou A, Sauter M, Hennenlotter    J, Wernet D, Stenzl A, Rammensee H G, Klingel K, Stevanović S.    Unexpected abundance of HLA class II presented peptides in primary    renal cell carcinomas. Clin Cancer Res. 2006 Jul. 15; 12(14 Pt    1):4163-70.-   Tran E, Turcotte S, Gros A, Robbins P F, Lu Y C, Dudley M E,    Wunderlich J R, Somerville R P, Hogan K, Hinrichs C S, Parkhurst M    R, Yang J C, Rosenberg S A. Cancer immunotherapy based on    mutation-specific CD4+ T cells in a patient with epithelial cancer.    Science. 2014 May 9; 344(6184):641-5.-   Singh-Jasuja H, Emmerich N P, Rammensee H G. The Tübingen approach:    identification, selection, and validation of tumor-associated HLA    peptides for cancer therapy. Cancer Immunol Immunother. 2004 March;    53(3):187-95. Epub 2004 Jan. 31. Review.-   Mori M, Beatty P G, Graves M, Boucher K M, Milford E L. HLA gene and    haplotype frequencies in the North American population: the National    Marrow Donor Program Donor Registry. Transplantation. 1997 Oct. 15;    64(7):1017-27.-   Colombetti S, Fagerberg T, Baumgartner P, Chapatte L, Speiser D E,    Rufer N, Michielin O, Lévy F. Impact of orthologous melan-A peptide    immunizations on the anti-self melan-A/HLA-A2 T cell    cross-reactivity. J Immunol. 2006 Jun. 1; 176(11):6560-7.-   Appay V, Speiser D E, Rufer N, Reynard S, Barbey C, Cerottini J C,    Leyvraz S, Pinilla C, Romero P. Decreased specific CD8+ T cell    cross-reactivity of antigen recognition following vaccination with    Melan-A peptide. Eur J Immunol. 2006 July; 36(7):1805-14.-   Xing Y, Hogquist K A. T-cell tolerance: central and peripheral. Cold    Spring Harb Perspect Biol. 2012 Jun. 1; 4(6).-   Aleksic M, Liddy N, Molloy P E, Pumphrey N, Vuidepot A, Chang K M,    Jakobsen B K. Different affinity windows for virus and    cancer-specific T-cell receptors: implications for therapeutic    strategies. Eur J Immunol. 2012 December; 42(12):3174-9.-   Burton E C, Prados M D. Malignant gliomas. Curr Treat Options Oncol.    2000 December; 1(5):459-68. Review.-   Dutoit V, Herold-Mende C, Hilf N, Schoor O, Beckhove P, Bucher J,    Dorsch K, Flohr S, Fritsche J, Lewandrowski P, Lohr J, Rammensee H    G, Stevanovic S, Trautwein C, Vass V, Walter S, Walker P R,    Weinschenk T, Singh-Jasuja H, Dietrich P Y. Exploiting the    glioblastoma peptidome to discover novel tumour-associated antigens    for immunotherapy. Brain. 2012 April; 135(Pt 4):1042-54.-   Tomita, Y., Harao, M., Senju, S., Imai, K., Hirata, S., Irie, A.,    Inoue, M., Hayashida, Y., Yoshimoto, K., Shiraishi, K., Mori, T.,    Nomori, H., Kohrogi, H. and Nishimura, Y. (2011), Peptides derived    from human insulin-like growth factor-II mRNA binding protein 3 can    induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes    reactive to cancer cells. Cancer Science, 102: 71-78.-   Barton V N, Donson A M, Birks D K, Kleinschmidt-DeMasters B K,    Handler M H, Foreman N K, Rush S Z (2013). Insulin-like growth    factor 2 mRNA binding protein 3 expression is an independent    prognostic factor in pediatric pilocytic and pilomyxoid astrocytoma.    J Neuropathol. Exp. Neurol. 72, 442-449.-   Beljan P R, Durdov M G, Capkun V, Ivcevic V, Pavlovic A, Soljic V,    Peric M (2012). IMP3 can predict aggressive behaviour of lung    adenocarcinoma. Diagn. Pathol. 7, 165.-   Bell J L, Wachter K, Muhleck B, Pazaitis N, Kohn M, Lederer M,    Huttelmaier S (2013). Insulin-like growth factor 2 mRNA-binding    proteins (IGF2BPs): post-transcriptional drivers of cancer    progression? Cell Mol. Life Sci. 70, 2657-2675.-   Chen C L, Tsukamoto H, Liu J C, Kashiwabara C, Feldman D, Sher L,    Dooley S, French S W, Mishra L, Petrovic L, Jeong J H, Machida K    (2013a). Reciprocal regulation by TLR4 and TGF-beta in    tumor-initiating stem-like cells. J Clin Invest 123, 2832-2849.-   Chen L T, Lin L J, Zheng L L (2013b). The correlation between    insulin-like growth factor II mRNA binding protein 3 expression in    hepatocellular carcinoma and prognosis. Hepatogastroenterology 60,    553-556.-   Chen P, Wang S J, Wang H B, Ren P, Wang X Q, Liu W G, Gu W L, Li D    Q, Zhang T G, Zhou C J (2012). The distribution of IGF2 and IMP3 in    osteosarcoma and its relationship with angiogenesis. J Mol. Histol.    43, 63-70.-   Chen S T, Jeng Y M, Chang C C, Chang H H, Huang M C, Juan H F, Hsu C    H, Lee H, Liao Y F, Lee Y L, Hsu W M, Lai H S (2011). Insulin-like    growth factor II mRNA-binding protein 3 expression predicts    unfavorable prognosis in patients with neuroblastoma. Cancer Sci.    102, 2191-2198.-   Chen Y L, Jeng Y M, Hsu H C, Lai H S, Lee P H, Lai P L, Yuan R H    (2013c). Expression of insulin-like growth factor II mRNA-binding    protein 3 predicts early recurrence and poor prognosis in    intrahepatic cholangiocarcinoma. Int. J Surg. 11, 85-91.-   Chiste M, Alexis J, Recine M (2014). IMP3 expression in serous    tumors of the ovary. Appl. Immunohistochem. Mol. Morphol. 22,    658-662.-   Del G A, Vaira V, Rocco E G, Palleschi A, Bulfamante G, Ricca D,    Fiori S, Bosari S, Ferrero S (2014). The Oncofetal Protein IMP3: A    Useful Marker to Predict Poor Clinical Outcome in Neuroendocrine    Tumors of the Lung. J Thorac. Oncol.-   Eldai H, Periyasamy S, Al Q S, Al R M, Muhammed M S, Deeb A, Al S E,    Afzal M, Johani M, Yousef Z, Aziz M A (2013). Novel genes associated    with colorectal cancer are revealed by high resolution cytogenetic    analysis in a patient specific manner. PLoS. ONE. 8, e76251.-   Feng W, Zhou Z, Peters J H, Khoury T, Zhai Q, Wei Q, Truong C D,    Song S W, Tan D (2011). Expression of insulin-like growth factor II    mRNA-binding protein 3 in human esophageal adenocarcinoma and its    precursor lesions. Arch. Pathol. Lab Med. 135, 1024-1031.-   Findeis-Hosey J J, Xu H (2012). Insulin-like growth factor    II-messenger RNA-binding protein-3 and lung cancer. Biotech.    Histochem. 87, 24-29.-   Gu L, Shigemasa K, Ohama K (2004). Increased expression of IGF II    mRNA-binding protein 1 mRNA is associated with an advanced clinical    stage and poor prognosis in patients with ovarian cancer. Int. J    Oncol 24, 671-678.-   Hammer N A, Hansen T, Byskov A G, Rajpert-De M E, Grondahl M L,    Bredkjaer H E, Wewer U M, Christiansen J, Nielsen F C (2005).    Expression of IGF-II mRNA-binding proteins (IMPs) in gonads and    testicular cancer. Reproduction. 130, 203-212.-   Hazama S, Nakamura Y, Takenouchi H, Suzuki N, Tsunedomi R, Inoue Y,    Tokuhisa Y, Iizuka N, Yoshino S, Takeda K, Shinozaki H, Kamiya A,    Furukawa H, Oka M (2014). A phase I study of combination vaccine    treatment of five therapeutic epitope-peptides for metastatic    colorectal cancer; safety, immunological response, and clinical    outcome. J Transl. Med. 12, 63.-   Hoffmann N E, Sheinin Y, Lohse C M, Parker A S, Leibovich B C, Jiang    Z, Kwon E D (2008). External validation of IMP3 expression as an    independent prognostic marker for metastatic progression and death    for patients with clear cell renal cell carcinoma. Cancer 112,    1471-1479.-   Hu S, Wu X, Zhou B, Xu Z, Qin J, Lu H, Lv L, Gao Y, Deng L, Yin J,    Li G (2014). IMP3 combined with CD44s, a novel predictor for    prognosis of patients with hepatocellular carcinoma. J Cancer Res    Clin Oncol 140, 883-893.-   Hwang Y S, Park K K, Cha I H, Kim J, Chung W Y (2012a). Role of    insulin-like growth factor-II mRNA-binding protein-3 in invadopodia    formation and the growth of oral squamous cell carcinoma in athymic    nude mice. Head Neck 34, 1329-1339.-   Hwang Y S, Xianglan Z, Park K K, Chung W Y (2012b). Functional    invadopodia formation through stabilization of the PDPN transcript    by IMP-3 and cancer-stromal crosstalk for PDPN expression.    Carcinogenesis 33, 2135-2146.-   Iinuma H, Fukushima R, Inaba T, Tamura J, Inoue T, Ogawa E, Horikawa    M, Ikeda Y, Matsutani N, Takeda K, Yoshida K, Tsunoda T, Ikeda T,    Nakamura Y, Okinaga K (2014). Phase I clinical study of multiple    epitope peptide vaccine combined with chemoradiation therapy in    esophageal cancer patients. J Transl. Med. 12, 84.-   Ikenberg K, Fritzsche F R, Zuerrer-Haerdi U, Hofmann I, Hermanns T,    Seifert H, Muntener M, Provenzano M, Sulser T, Behnke S, Gerhardt J,    Mortezavi A, Wild P, Hofstadter F, Burger M, Moch H, Kristiansen G    (2010). Insulin-like growth factor II mRNA binding protein 3 (IMP3)    is overexpressed in prostate cancer and correlates with higher    Gleason scores. BMC. Cancer 10, 341.-   Jeng Y M, Chang C C, Hu F C, Chou H Y, Kao H L, Wang T H, Hsu H C    (2008). RNA-binding protein insulin-like growth factor II    mRNA-binding protein 3 expression promotes tumor invasion and    predicts early recurrence and poor prognosis in hepatocellular    carcinoma. Hepatology 48, 1118-1127.-   Jeng Y M, Wang T H, Lu S H, Yuan R H, Hsu H C (2009). Prognostic    significance of insulin-like growth factor II mRNA-binding protein 3    expression in gastric adenocarcinoma. Br. J Surg 96, 66-73.-   Jin L, Seys A R, Zhang S, Erickson-Johnson M R, Roth C W, Evers B R,    Oliveira A M, Lloyd R V (2010). Diagnostic utility of IMP3    expression in thyroid neoplasms: a quantitative RT-PCR study. Diagn.    Mol. Pathol. 19, 63-69.-   Kazeminezhad B, Mirafsharieh S A, Dinyari K, Azizi D, Ebrahimi A    (2014). Usefulness of insulin-like growth factor II mRNA-binding    protein 3 (IMP3) as a new marker for the diagnosis of esophageal    adenocarcinoma in challenging cases. Turk. J Gastroenterol. 25,    253-256.-   Kleinschmidt-DeMasters B K, Donson A M, Vogel H, Foreman N K (2014).    Pilomyxoid Astrocytoma (PMA) Shows Significant Differences in Gene    Expression vs. Pilocytic Astrocytoma (PA) and Variable Tendency    Toward Maturation to PA. Brain Pathol.-   Kobel M, Xu H, Bourne P A, Spaulding B O, Shih I, Mao T L, Soslow R    A, Ewanowich C A, Kalloger S E, Mehl E, Lee C H, Huntsman D, Gilks C    B (2009). IGF2BP3 (IMP3) expression is a marker of unfavorable    prognosis in ovarian carcinoma of clear cell subtype. Mod. Pathol.    22, 469-475.-   Kono K, linuma H, Akutsu Y, Tanaka H, Hayashi N, Uchikado Y, Noguchi    T, Fujii H, Okinaka K, Fukushima R, Matsubara H, Ohira M, Baba H,    Natsugoe S, Kitano S, Takeda K, Yoshida K, Tsunoda T, Nakamura Y    (2012). Multicenter, phase II clinical trial of cancer vaccination    for advanced esophageal cancer with three peptides derived from    novel cancer-testis antigens. J Transl. Med. 10, 141.-   Kono K, Mizukami Y, Daigo Y, Takano A, Masuda K, Yoshida K, Tsunoda    T, Kawaguchi Y, Nakamura Y, Fujii H (2009). Vaccination with    multiple peptides derived from novel cancer-testis antigens can    induce specific T-cell responses and clinical responses in advanced    esophageal cancer. Cancer Sci. 100, 1502-1509.-   Li D, Yan D, Tang H, Zhou C, Fan J, Li S, Wang X, Xia J, Huang F,    Qiu G, Peng Z (2009). IMP3 is a novel prognostic marker that    correlates with colon cancer progression and pathogenesis. Ann Surg    Oncol 16, 3499-3506.-   Li H G, Han J J, Huang Z Q, Wang L, Chen W L, Shen X M (2011). IMP3    is a novel biomarker to predict metastasis and prognosis of tongue    squamous cell carcinoma. J Craniofac. Surg. 22, 2022-2025.-   Liao B, Hu Y, Brewer G (2011). RNA-binding protein insulin-like    growth factor mRNA-binding protein 3 (IMP-3) promotes cell survival    via insulin-like growth factor II signaling after ionizing    radiation. J Biol. Chem. 286, 31145-31152.-   Liao B, Hu Y, Herrick D J, Brewer G (2005). The RNA-binding protein    IMP-3 is a translational activator of insulin-like growth factor II    leader-3 mRNA during proliferation of human K562 leukemia cells. J    Biol. Chem. 280, 18517-18524.-   Lin L, Zhang J, Wang Y, Ju W, Ma Y, Li L, Chen L (2013).    Insulin-like growth factor-II mRNA-binding protein 3 predicts a poor    prognosis for colorectal adenocarcinoma. Oncol Lett. 6, 740-744.-   Liu H, Shi J, Anandan V, Wang H L, Diehl D, Blansfield J, Gerhard G,    Lin F (2012). Reevaluation and identification of the best    immunohistochemical panel (pVHL, Maspin, 5100P, IMP-3) for ductal    adenocarcinoma of the pancreas. Arch. Pathol. Lab Med. 136, 601-609.-   Lochhead P, Imamura Y, Morikawa T, Kuchiba A, Yamauchi M, Liao X,    Qian Z R, Nishihara R, Wu K, Meyerhardt J A, Fuchs C S, Ogino S    (2012). Insulin-like growth factor 2 messenger-   RNA binding protein 3 (IGF2BP3) is a marker of unfavourable    prognosis in colorectal cancer. Eur. J Cancer.-   Lu D, Vohra P, Chu P G, Woda B, Rock K L, Jiang Z (2009). An    oncofetal protein IMP3: a new molecular marker for the detection of    esophageal adenocarcinoma and high-grade dysplasia. Am J Surg    Pathol. 33, 521-525.-   Lu D, Yang X, Jiang N Y, Woda B A, Liu Q, Dresser K, Mercurio A M,    Rock K L, Jiang Z (2011). IMP3, a new biomarker to predict    progression of cervical intraepithelial neoplasia into invasive    cancer. Am. J Surg. Pathol. 35, 1638-1645.-   Mizukami Y, Kono K, Daigo Y, Takano A, Tsunoda T, Kawaguchi Y,    Nakamura Y, Fujii H (2008). Detection of novel cancer-testis    antigen-specific T-cell responses in TIL, regional lymph nodes, and    PBL in patients with esophageal squamous cell carcinoma. Cancer Sci.-   Morimatsu K, Aishima S, Yamamoto H, Hayashi A, Nakata K, Oda Y,    Shindo K, Fujino M, Tanaka M, Oda Y (2013). Insulin-like growth    factor II messenger RNA-binding protein-3 is a valuable diagnostic    and prognostic marker of intraductal papillary mucinous neoplasm.    Hum. Pathol. 44, 1714-1721.-   Nielsen J, Christiansen J, Lykke-Andersen J, Johnsen A H, Wewer U M,    Nielsen F C (1999). A family of insulin-like growth factor II    mRNA-binding proteins represses translation in late development. Mol    Cell Biol. 19, 1262-1270.-   Noske A, Faggad A, Wirtz R, Darb-Esfahani S, Sehouli J, Sinn B,    Nielsen F C, Weichert W, Buckendahl A C, Roske A, Muller B, Dietel    M, Denkert C (2009). IMP3 expression in human ovarian cancer is    associated with improved survival. Int. J Gynecol. Pathol. 28,    203-210.-   Okada K, Fujiwara Y, Nakamura Y, Takiguchi S, Nakajima K, Miyata H,    Yamasaki M, Kurokawa Y, Takahashi T, Mori M, Doki Y (2012).    Oncofetal protein, IMP-3, a potential marker for prediction of    postoperative peritoneal dissemination in gastric adenocarcinoma. J    Surg. Oncol 105, 780-785.-   Pryor J G, Bourne P A, Yang Q, Spaulding B O, Scott G A, Xu H    (2008). IMP-3 is a novel progression marker in malignant melanoma.    Mod. Pathol. 21, 431-437.-   Rammensee H G, Bachmann J, Emmerich N P, Bachor O A, Stevanovic S    (1999). SYFPEITHI: database for MHC ligands and peptide motifs.    Immunogenetics 50, 213-219.-   Rammensee H G, Bachmann J, Stevanovic S (1997). MHC Ligands and    Peptide Motifs. (Heidelberg, Germany: Springer-Verlag).-   Rivera V T, Boudoukha S, Simon A, Souidi M, Cuvellier S, Pinna G,    Polesskaya A (2013). Post-transcriptional regulation of cyclins D1,    D3 and G1 and proliferation of human cancer cells depend on IMP-3    nuclear localization. Oncogene.-   Samanta S, Sharma V M, Khan A, Mercurio A M (2012). Regulation of    IMP3 by EGFR signaling and repression by ERbeta: implications for    triple-negative breast cancer. Oncogene 31, 4689-4697.-   Samanta S, Sun H, Goel H L, Pursell B, Chang C, Khan A, Greiner D L,    Cao S, Lim E, Shultz L D, Mercurio A M (2015). IMP3 promotes    stem-like properties in triple-negative breast cancer by regulating    SLUG. Oncogene.-   Schaeffer D F, Owen D R, Lim H J, Buczkowski A K, Chung S W,    Scudamore C H, Huntsman D G, Ng S S, Owen D A (2010). Insulin-like    growth factor 2 mRNA binding protein 3 (IGF2BP3) overexpression in    pancreatic ductal adenocarcinoma correlates with poor survival. BMC.    Cancer 10, 59.-   Serao N V, Delfino K R, Southey B R, Beever J E, Rodriguez-Zas S L    (2011). Cell cycle and aging, morphogenesis, and response to stimuli    genes are individualized biomarkers of glioblastoma progression and    survival. BMC. Med. Genomics 4, 49.-   Su P, Hu J, Zhang H, Li W, Jia M, Zhang X, Wu X, Cheng H, Xiang L,    Zhou G (2014). IMP3 expression is associated with    epithelial-mesenchymal transition in breast cancer. Int. J Clin Exp.    Pathol. 7, 3008-3017.-   Suda T, Tsunoda T, Daigo Y, Nakamura Y, Tahara H (2007).    Identification of human leukocyte antigen-A24-restricted epitope    peptides derived from gene products upregulated in lung and    esophageal cancers as novel targets for immunotherapy. Cancer Sci.-   Suvasini R, Shruti B, Thota B, Shinde S V, Friedmann-Morvinski D,    Nawaz Z, Prasanna K V, Thennarasu K, Hegde A S, Arivazhagan A,    Chandramouli B A, Santosh V, Somasundaram K (2011). Insulin growth    factor-2 binding protein 3 (IGF2BP3) is a glioblastoma-specific    marker that activates phosphatidylinositol    3-kinase/mitogen-activated protein kinase (PI3K/MAPK) pathways by    modulating IGF-2. J Biol. Chem. 286, 25882-25890.-   Szarvas T, Tschirdewahn S, Niedworok C, Kramer G, Sevcenco S, Reis    H, Shariat S F, Rubben H, Vom D F (2014). Prognostic value of tissue    and circulating levels of IMP3 in prostate cancer. Int. J Cancer    135, 1596-1604.-   Takata A, Takiguchi S, Okada K, Takahashi T, Kurokawa Y, Yamasaki M,    Miyata H, Nakajima K, Mori M, Doki Y (2014). Expression of    insulin-like growth factor-II mRNA-binding protein-3 as a marker for    predicting clinical outcome in patients with esophageal squamous    cell carcinoma. Oncol Lett. 8, 2027-2031.-   Taniuchi K, Furihata M, Hanazaki K, Saito M, Saibara T (2014).    IGF2BP3-mediated translation in cell protrusions promotes cell    invasiveness and metastasis of pancreatic cancer. Oncotarget. 5,    6832-6845.-   Tomita Y, Harao M, Senju S, Imai K, Hirata S, Irie A, Inoue M,    Hayashida Y, Yoshimoto K, Shiraishi K, Mori T, Nomori H, Kohrogi H,    Nishimura Y (2011). Peptides derived from human insulin-like growth    factor-II mRNA binding protein 3 can induce human leukocyte    antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer    cells. Cancer Sci. 102, 71-78.-   Ueki A, Shimizu T, Masuda K, Yamaguchi S I, Ishikawa T, Sugihara E,    Onishi N, Kuninaka S, Miyoshi K, Muto A, Toyama Y, Banno K, Aoki D,    Saya H (2012). Up-regulation of Imp3 confers in vivo tumorigenicity    on murine osteosarcoma cells. PLoS. ONE. 7, e50621.-   Vikesaa J, Hansen T V, Jonson L, Borup R, Wewer U M, Christiansen J,    Nielsen F C (2006). RNA-binding IMPs promote cell adhesion and    invadopodia formation. EMBO J 25, 1456-1468.-   Wachter D L, Kristiansen G, Soil C, Hellerbrand C, Breuhahn K,    Fritzsche F, Agaimy A, Hartmann A, Riener M O (2012). Insulin-like    growth factor II mRNA-binding protein 3 (IMP3) expression in    hepatocellular carcinoma. A clinicopathological analysis with    emphasis on diagnostic value. Histopathology 60, 278-286.-   Wachter D L, Schlabrakowski A, Hoegel J, Kristiansen G, Hartmann A,    Riener M O (2011). Diagnostic value of immunohistochemical IMP3    expression in core needle biopsies of pancreatic ductal    adenocarcinoma. Am. J Surg. Pathol. 35, 873-877.-   Wang B J, Wang L, Yang S Y, Liu Z J (2015). Expression and clinical    significance of IMP3 in microdissected premalignant and malignant    pancreatic lesions. Clin Transl. Oncol 17, 215-222.-   Wang L, Li H G, Xia Z S, Lu J, Peng T S (2010). IMP3 is a novel    biomarker to predict metastasis and prognosis of gastric    adenocarcinoma: a retrospective study. Chin Med. J (Engl.) 123,    3554-3558.-   Wang Y, Li L, Wang Y, Yuan Z, Zhang W, Hatch K D, Zheng W (2014).    IMP3 as a cytoplasmic biomarker for early serous tubal    carcinogenesis. J Exp. Clin Cancer Res. 33, Yantiss R K, Cosar E,    Fischer A H (2008). Use of IMP3 in identification of carcinoma in    fine needle aspiration biopsies of pancreas. Acta Cytol. 52,    133-138.-   Yoshino K, Motoyama S, Koyota S, Shibuya K, Sato Y, Sasaki T, Wakita    A, Saito H, Minamiya Y, Sugiyama T, Ogawa J (2014). Identification    of insulin-like growth factor 2 mRNA-binding protein 3 as a    radioresistance factor in squamous esophageal cancer cells. Dis.    Esophagus. 27, 479-484.-   Yuan R, Chen Y, He X, Wu X, Ke J, Zou Y, Cai Z, Zeng Y, Wang L, Wang    J, Fan X, Wu X, Lan P (2013). CCL18 as an independent favorable    prognostic biomarker in patients with colorectal cancer. J Surg. Res    183, 163-169.-   Yuan R H, Wang C C, Chou C C, Chang K J, Lee P H, Jeng Y M (2009).    Diffuse expression of RNA-binding protein IMP3 predicts high-stage    lymph node metastasis and poor prognosis in colorectal    adenocarcinoma. Ann Surg Oncol 16, 1711-1719.-   Zhang Y, Garcia-Buitrago M T, Koru-Sengul T, Schuman S, Ganjei-Azar    P (2013). An immunohistochemical panel to distinguish ovarian from    uterine serous papillary carcinomas. Int. J Gynecol. Pathol. 32,    476-481.

The invention claimed is:
 1. A method of treating a patient who hascancer, comprising administering to the patient an effective amount ofan antibody specifically binding to an MHC class I or II moleculecomplexed with a HLA-restricted antigen consisting of the amino acidsequence of KIQEILTQV (SEQ ID NO: 1), wherein said cancer is selectedfrom the group consisting of oral carcinomas, H. pylori-induced MALTlymphoma, Ewing's sarcoma, head and neck squamous cell carcinoma,epithelial cancer of the larynx, atypical meningioma, papillary thyroidcarcinoma, salivary duct carcinoma, cervical cancer, and extranodalT/NK-cell lymphomas.
 2. The method of claim 1, wherein the antibody is apolyclonal antibody, a monoclonal antibody, or a chimeric antibody. 3.The method of claim 1, wherein the antibody binds to the HLA-restrictedantigen with a binding affinity of below 20 nanomolar.
 4. The method ofclaim 1, wherein the antibody binds to the MEW class I moleculecomplexed with the HLA-restricted antigen.
 5. The method of claim 1,wherein the antibody is humanized.
 6. The method of claim 1, wherein theeffective amount of the antibody is from about 1 μg/kg to about 100mg/kg of body weight per day.
 7. The method of claim 1, wherein theantibody is conjugated with a toxin.
 8. The method of claim 1, whereinthe antibody is conjugated with an immune stimulating domain.
 9. Themethod of claim 1, wherein the cancer is cervical cancer.
 10. The methodof claim 1, wherein the cancer is oral carcinomas.
 11. The method ofclaim 1, wherein the cancer is Ewing's sarcoma.
 12. The method of claim1, wherein the cancer is head and neck squamous cell carcinoma.
 13. Themethod of claim 1, wherein the cancer is epithelial cancer of thelarynx.
 14. The method of claim 1, wherein the cancer is salivary ductcarcinoma.
 15. The method of claim 1, wherein the cancer is extranodalT/NK-cell lymphomas.